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Regenerative Medicine > Application of Mammalian Cells > Flashcards

Application of Mammalian Cells Flashcards

1
Q

What is an antibody?

A

A naturally occurring protein used by the immune system to identify and neutralise foreign objects (eg bacteria, viruses)

Each antibody recognises a specific antigen unique to its target

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2
Q

What is an antigen?

A

Any substance that causes the immune system to produce antibodies against it

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3
Q

What is an epitope?

A

The specific region in the antigen that is recognised by the immune cells

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4
Q

Name the two antibody types and explain them

A

Monoclonal (MAb)
- identical antibodies produced by one type of immune cell
- expensive can be produced in large quantities - infinite supply
- bind to same epitopes on taerget antigen

Polyclonal
- derived from different cell lines, mixture of antibodies
- cheaper but limited supply
- bind to same antigen but different epitopes

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5
Q

What are the 2 major functions of an antibodies?

A

1) to recognise and bind to antigens

2) to induce immune response after binding

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6
Q

What are the regions of an antibody and what do they do ?

A

The variable region mediates binding

The affinity for a given antigen is determined by the variable region

The constant region mediates the immune response after binding

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7
Q

What are the historical milestones associated with antibodies?

A

1975 - cesar milstein and George’s kohler : the hybridoma technique

1984 - milatein and kohler received Nobel prize

1986 - first mouse monoclonal antibody approved for human use ( murimonav - cd3- immunosuppressant for organ transplant)

2003 - first human monoclonal antibody (adalimumab = humira- for rheumatoid arthritis

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8
Q

Explain the invivo and in vitro method of the hybridoma technique

A

In vivo : injected in mice ( in peritoneal cavity, gut) produce tumours containing antibodies rich fluid (ascites fluid)

In vitro : grown indefinitely in cell culture media

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9
Q

Give the advantages and disadvantages of the in vivo method

A

ADV
- cheaper
- higher MAb production yield (5-20mg/ml)

DISADV
- unethical
- requires specialised personnel (specialised license for handling animals

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10
Q

Give the advantages and disadvantages of the in vitro method

A

ADV
- ethical
- doesn’t require specialised personnel

DISADV
- lower MAb productivity (5-10ug/ml)
- some hybridoma lines don’t grow in culture

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11
Q

Why are hybridoma cells micro encapsulated in alginate capsules?

A

This is because it significantly increases the MAb yields (10-100ug/ml) as a result of higher cell density

Damon biotech company and cell tech use encapsulated hybridoma cells for large scale production of MAbs. They employ 100 litre fermenters to yield about 100g of MAbs in about 2 week period

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12
Q

What are the disadvantages of the hybridoma technique?

A
  • laborious
  • expensive
  • time consuming
  • often caused immune reactions in patients
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13
Q

Suggest an alternative technology for MAb production

A

Recombinant DNA Technology

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14
Q

What is recombinant DNA technology ?

A

The insertion of DNA molecules/particles from a different species into a host organism (expression system) to produce useful products

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15
Q

Explain the basic principles of recombinant dna technology

A

When a fragment of dna from a donor cell or organism is isolated and then inserted into the dna of another cell or organism

This allows scientists Toni traduce a new characteristic into and organism by inserting a new gene into it

Recombinant dna tech involves 4 steps
1) a dna fragment containing the gene of interest is obtained from donor cell
2) a suitable plasmid is obtained from a bacterium
(Plasmids are commonly used as vectors to transfer the gene of interest into the host cell for expression
3) the dna fragment containing the gene of interest is cut using an enzym called restriction enzyme (enzyme recognises a specific base sequence and cures the dna at a specific point)
Same restriction enzyme is used to cut the plasmid
4) the dna fragment containing th green of interest is inserted into the open plasmid with the help of another enzyme called dna ligase (acts like a glue)
Calatlyses the jointing of dna fragment and plasmid

A recombinant plasmid is formed
The plasmid can be introduced to a host cell for different purposes for example producing protein or GM fruit and veg

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16
Q

Give 3 examples of expression vectors

A
  • plasmid DNA
  • virus vector
  • bacterial vector
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17
Q

Give 2 examples of expression systems

A

Prokaryotic - bacteria ( ecoli)

Eukaryotic
- yeast ( sacharomyces verrvisiae)
- viral (baculovirus)
- mammalian ( CHO)

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18
Q

What expression system would be used for the following applications : large protein, small proteins, glycosylation, high yield low cost and post translational modifications?

A

Large proteins (>100 KD ) - eukaryote

Small protein (<30kD) - prokaryote

Glycosylation - baculovirus or mammalian cell culture

High yield low cost - E. coli

Post transitional modifications - yeast or baculovirus or mammalian cells

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19
Q

What are the advantages and disadvantages of mammalian cells (eg CHO, HEK293) as expression systems?

A

Adv
- increased level of expression
- production of native structure proteins (correctly folded with appropriate bonding)
- easy to scale up by fermentation

Disadv
- expensive
- slower growing

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20
Q

What are the advantages and disadvantages of bacterial cells (eg E.coli) as expression systems?

A

Adv
- Simple, well understood genetics
- easy to manipulate genetically
- very cheap
- fast expression with short doubling time
- easy to scale up by fermentation

Disadv
- protein folding issue resulting in insoluble inclusion bodies
- low yield for large proteins
- glycoprotein modification issues

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21
Q

What is transformation in terms of DNA?

A

The alteration of genetic composition of a cell via the uptake of foreign DNA
- naturally I’m bacteria bu conjunction ( intra or inter species)
- naturally in bacteria by transduction ( viral- medicated DNA incorporation)
- artificially by chemical method and by electroporation

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22
Q

What is transfection?

A

The transformation and infection of cells to introduce foreign DNA

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23
Q

List 6 properties of transient transfection

A
  • No genomic integration
  • short term expression (24-48h)
  • DNA is degraded by nucleuses or dilutes during cell division
  • useful for rapid analysis of phenotype or for recombinant protein production
  • works best with supercooled circular plasmids
  • chemical method or electroporation
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24
Q

List 6 properties of stable transfection

A

DNA is integrated into the cells genome and is replicated with the host DNA

Permanent

Sustained expression for long periods of time

Works best with viral DNA

Viral or micronisation

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25
Q

Explain the process of transfection

A

Rely on masking the negatuve charges of the phosphate backbone of DNA to facilitate precipitation/ cell uptake

Particulate DNA complexes are taken up into cells by endocytosis

Naked DNA is digested within minute in the cytosol by nucleases

DNA is delivered to the nucleus by unknown mechanisms

Not clear whether DNA is delivered into the nucleus as a particulate (protected) or naked

Methods:
- calcium phosphate
- positively charged polymers
- liposome

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26
Q

What are the main 3 chemical methods of transfections?

A

Methods:
- calcium phosphate
- positively charged polymers
- liposome

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27
Q

Explain the calcium phosphate transfection

A

Use calcium phosphate to precipitate the DNA

Precipitates DNA added to cell media and eventually the cells will uptake it by endocytosis

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28
Q

What are the advantages and disadvantages of calcium phosphate transfection?

A

Advantages
- very simple
- inexpensive
- we’ll established
- works for most cell types

Disadvantages
- works for adherent cells only
- skill and experience required to get good precipitates
- not particularly efficient in some cell types
- many cells types do not like adherent precipitates
- difficult to automate or perform as a high throughput method

29
Q

How is transfection done using positively charged polymers?

A

Polymer DIETHTLAMINOETHYL modified dextran used

DNA adheres to the DEAE dextran and remains soluble

The complex adheres to cells and is taken up by endocytosis

30
Q

What are the advantages and disadvantages of transfection using positively charged polymers?

A

Advantages
- gentle, low toxicity
- works well for cells in suspension
- high volume transfection

Disadvantages
- doesn’t work well I many cell types
- doesn’t work well for stable transfection
- unclear mechanism of action makes optimisation difficult
- relatively expensive
- low throughput

31
Q

What are liposomes?

A

Liposomes are vesicle composed of a lipid bilayer surrounding a hollow core into which drugs or other molecules can be loaded for delivery

32
Q

How are unilamellar liposomes formed?

A

Can be formed from cationic lipids by :
Sonication - uneven liposome size, not reproducible
Microfluidisation - regular, small, stable liposomes, reproducible

33
Q

How does liposomes mediated transfection work?

A

DNA is mixed with liposomes and binds electrostatically

Liposome/DNA complexes enter cells by fusion with plasma

Encapsulate gene in liposome which then fuses with plasma membrane and uptakes through endocytosis by cell

34
Q

What are the advantages and disadvantages of liposome mediated transfection?

A

Advantages
- very simple to perform and optimise - anyone can do it
- easy to automate, high throughput
- reliable and reproducible
- stable and transient assays work well
- works well with many cell types and also in vivo, either adherent or non adherent cells

Disadvantages
- many formulations require use of serum free or serum reduce medium for good efficiency ( all types of neutral lipids)
- some formulations are unstable to oxygen
- variable toxicity necessitates carful optimisation for many types ( eg lypofectamine)
- very expensive to but ( but almost free to make)

35
Q

What is electroporation?

A

Uses electrical field that transiently permeabilises the membrane during this period, the foreigner material can enter the cells

36
Q

How is electroporation carried out?

A

Cells and DNA mixed and places into a cuvette between two electrode plates

High dc voltage applied as a pulse

Optimised by altering voltage, pulse length, wave form.

During electroporation electro pulse applied creating gaps in cell membrane, temporary, through pore DNA can pass through

Dna suspended in conductive solution
Close circuit
Short pulse lasting milliseconds applied distrusting bilayer
As electric potential across cell membrane rises due to the voltage applied allows charged particles to pass through

37
Q

What are the advantages and disadvantages of electroporation ( non chemical transfection methods)?

A

Advantages
- high transfection efficiency
- effective for stable transfection

Disadvantages
- only works for cells in suspension
- devices for transferring adherent cells are tricky and cumbersome to clean
- required optimisation as cell permeability can lead to cell damages
- experienced users are required
- very sensitive to salt concentrations
- expensive

38
Q

How is viral transfection or transduction carried out?

A

Happen naturally when virus transfers genetic material from one bacteria to another

Transferring foreign DNA into a cell using a virus

39
Q

When and why is viral transfection used and talk about it’s efficiency

A
  • used for hart to transfer cell types
  • commonly used in clinical research
  • often 100% transfection efficiency
  • viruses evolved to efficiently introduce nuclei acid into cells
  • can be used for in vivo transfection
  • engineered for maximum infection but replication defective by mutation / deletion of gag, pol, Env
40
Q

Give examples of viral transfection cells and why they’re used

A

Adenoviruses, retroviruses - used extensively for gene delivery in mammalian cell cultures

Baculovirus - expression in insect cells for Pepsi ting recombinant glycoproteins or membrane proteins

41
Q

What is a vaccine?

A

biological preparation that provide active acquired immunity to a particular disease

Often made from weakened, inactivated or killed forms of microbes, toxins or one of its surface proteins

42
Q

How is a virus produced?

A

Not very different from antibody production

The virus replicates inside cells and the cell environment is the ‘culture enviroment’

(Culture within a culture)

Viruses can be produced in eggs ( influenza) or in mammalian cell cultures

43
Q

How are vaccines created in embryo ages eggs?

A

1) Fertilised eggs held in incubators (9-11 days)

2) inculcation with specific influenza virus

3) incubation for 48196 hours at 33-36*c

4) pre chilling in refrigerator

5) harvest of allantonic fluid

6) purification and virus inactivation

(Approx 15ug viral vaccine produces from 1 or 2 eggs ( single human dose))

44
Q

Why are embryonated eggs used for vaccine production?

A

Eggs are perfect natural mini bioreactors ( nutrient rich, aseptic)

Many viruses grow very well in ebryonated chicken eggs

Robust yield enables use for reaserch and vaccine production

45
Q

Give 5 examples of vaccines and what cells they use

A

Flucelvax (us) / optaflu(eu) (Novartis) - first mammalian cell based vaccine against the influenza virus (2012) produced in MDCK cells

Polio - in vero cells or human diploid fibroblasts

Rabies - in vero cells

Measles, mumps - in chicken embryo cells

Rubella, chicken pox - in human diploid lung fibroblasts

46
Q

How are vaccines produced when creating it using a mammalian cell culture?

A

choose cell line of interest example - vero cells

Two phases
Growth phase - objective is to increase mammalian cell biomass once preferred cell density reached they are infected with viral particles

Production phase - objective is to maximise the conversion of the mammalian cell biomass to viable viral biomass

Disposable bioreactors usually used
2 types can be used either horizontal on orbital shakers or vertical bags have impeller in them to ensure good mixing

47
Q

What cell types is most suitable for viral growth for large scale productions?

A

Anchorage dependant cell growth with micro-carrier cultures often used

48
Q

Why use animal cell cultures for viral vaccine production?

A

Animal viruses propagated by cultured cells at relatively high density

49
Q

How is virus production maximised?

A

Viral inoculation is accompanied by a feeder to keep cells alive and maximise virus production

50
Q

What is polio?

A

Poliomyelitis or infantile paralysis

Caused by the polio virus

Transmission by oral faecal route

Multiplies in throat and intestine

Invaded blood and lymph

Sub clinical - mild symptoms in 95% of people

Clinical - non paralytic polio , paralytic polio 1% of cases

51
Q

Explain the properties of the polio virus and it’s associated timeline

A

2 types of vaccine
- an inactivated poliovirus given by injection
- a weakened poliovirus given by mouth

Developed and tested in 1952 released 1955

Cultured live polio virus in monkey kidney epithelial cells (vero)

Virus harvested and killed by formaldehyde

Injected vaccine
- provides igG mediated immunity to prevent viremia
- 60-90% protection

I’m 1957 Albert Savin Sabin developed a live attenuate polio vaccine by passaging poliovirus through monkeys
- oral vaccine that given intestinal mucosal immunity
- occasionally mutates back and gives people polio

52
Q

What’s an antibiotic?

A

Chemical non toxic compounds used to treat infections caused primarily by bacteria.

53
Q

What’s a natural antibiotic?

A

True antibiotic - produced naturally by fungi or bacteria to selectively inhibit the growth of others ( chrysogenum produces penicillin

54
Q

What’s a semi synthetic antibiotic?

A

Chemically modified natural antibiotics ( ampicillin)

55
Q

What’s a totally synthetic antibiotic?

A

Manufactured antibiotic like trimethoprim

56
Q

Describe the timeline for the discovery of penicillin

A

1928 : Alexander Fleming noticed a secreted anti microbial produced by the penicillium fungus. This compound was penicillin

Between 1928 and 1945 Howard Florey and Ernst chain devised methods to produce large quantities of penicillin - the first B-road spectrum antibiotic with low toxicity and very good activity

1940s : penicillin was first introduced at large scale (20l) and purified by Howard Florey and Ernst chain

Fleming chain and Florey shared the Nobel prize for medicine in 1945

57
Q

How do antibiotics work?

A

They work in one of 2 ways :
Bactericidal: kills the bacteria by preventing it to make a cell wall

Bacteriostatic : prevents bacteria from dividing by interfering with processing like DNA replication, metabolism ( enzyme activity) and protein production

58
Q

What are broad and narrow spectrum antibiotics?

A

Broad spectrum - kill a wide range of bacteria ( penicillin)

Narrow spectrum - kill a specific types or group of bacteria ( isoniazid)

59
Q

What are the 4 mechanism of action of antibiotics?

A

Cell wall synthesis inhibitors
- b lactams
- glycopeptides
- cycloserine, bacitracin

Targeting cell membrane
- polymyxin

Inhibit nucleic acid synthesis (DNA replication)
- sulphinamides
- inhibits cell division not kill

Inhibit protein synthesis
- stops replication
- tetracyclines

60
Q

What was antibiotic production like in 1945?

A

Mass production was possible

Yield was 1g/l of penicillin
Not great yield

Biggest discovery I’m medicine

61
Q

What does antibiotic production look like currently?

A

Same fungus used
Yields 50g/L of penicillin

Culture medium - lactose, other sugars, source of nitrogen - yeast extract

Fermented in fed batch mode ( to prolong stationary phase of the cells and increase production)

62
Q

What phase of the growth cycle does penicillin production occure?

A

Penicillin is a secondary metabolite so is produced during the stationary phase of cell growth

63
Q

Why is penicillin modified?

A

Penicillin G can be enzymatically modified to make a variety of other penicillins with slightly different properties ( penicillin O, ampicillin, amoxicillin)

Depending on what properties are required the penicillin is adjusted accordingly

64
Q

Explain the downstream bioprocessing of antibiotics m

A

Required to increase purity

1) involves filtration to remove solids

2) the filtered broths is acidified and extracted with an organic solvent
Extract is then stripped with buffer to isolate a concentrate

3) pH of concentrate is adjusted with acid and product is purified by second extraction into organic solvent

4) final polishing consists of vaccine concentration, crystallisation, and drying of crystalline penicillin

65
Q

What is cephalosporin?

A

A b lectim antibiotic used to treat wide range of infections from grim positive to grim negative bacteria

Broad spectrum antibiotic

66
Q

What are the culture conditions for celphalosporin?

A

Media - source carbon ( glucose, starch, sucrose), source of nitrogen (meat extract, peptone)

Growth conditions - pH 6-7 temp 24-28, for 4-10 days

High oxygen consumption - compressed filter sterilised air is supplied

Fermentation - fed batch mode to prolong stationary phase

Downstream
RIPP

67
Q

How does antibiotic resistance occur?

A

High number of bacteria
A few of them are resistant to antibiotics

Antibiotics kill bacteria causing illness as well as good bacteria protecting body from infection

The resistant bacteria now have preferred conditions to grow and take over

Bacteria can transfer their drug resistance to other bacteria causing more problems

68
Q

How long can it take to bring a new antibiotic onto the market?

A

Up to 15 years

69
Q

Explain the steps of the hybridoma technique for producing monoclonal antibodies

A

1) immunisation of mouse

2) B cell isolation from spleen

3) in parallel, the cultivation of myeloma cells ( lack the HGRT gene)

4) myeloma and B cells fusion ( electro fusion or chemically

5) culture fused in HAT medium for 10-14 days. Unfused myeloma cells will die in the HAT medium. Unfused B cells die because they have a short life span

6) dilution of the hybridoma suspension until one cell per well

7) cell line screening for antibodies of appropriate specificity ( by using elsa)

8) in vitro (a) or in vivo (b) multiplication

9) harvesting of MAbs

Regenerative Medicine (8 decks)

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